This project will examine the cellular and molecular mechanisms that govern the establishment and refinement of synaptic connections in a model system, the Drosophila neuromuscular junction. Recent studies in this system indicate that axon guidance and synaptogenesis involve multiple repulsive and attractive signals that act on the growth cones. The mechanisms of efferent outgrowth and neuromuscular taget selection will be examined in developing embryos and larvae using new Drosophila lines with endogenous Green Fluorescent Protein (GFP) expression. Detailed time-lapse morphometric studies of the growth cones will be made, testing the resonses to several cellular, genetic, and molecular manipulations. There is good evidence for morphological plasticity in the neuromuscular system, in response to alterations of the degree of innervation and synaptic activity. The mechanisms of motoneuron arbor refinement and plasticity will be tested by vital imaging of the developing synapses in both embros and lavae, in response to situations where neuromuscular activity is altered pharmacologically or genetically, as well as in response to denervation or altered innervation protocols. Also, candidate genes whose functions are needed to mediate neuromuscular development and synaptic plasticity will be identified in a F1 mutagenesis screen. Finally, as part of a revers genetic study, 4200 P-element enhancer detector inserts were screened for expression patterns in the developing neuromuscular system. 20 Drosophila lines were identified with specificity of expression in subsets of muscle fibers and/or neurons during embryonic synaptogenesis. We will complete an ongoing molecular characterization of these enhancer detector lines, including a detailed analysis of mutant phenotypes that result from excision of the P-element inserts.